As described in project 1, the highly conserved Wnt/beta-catenin signaling pathway is a crucial pathway for embryogenesis. Mutations in oncogenes or tumor suppressors in the Wnt/beta-catenin pathway lead to its constitutive activation and cause cancer in the adult. Alternative Wnt signaling pathways, controlled by noncanonical Wnts such as Wnt5a, have been shown in vitro to antagonize Wnt/beta-catenin signaling suggesting that stimulation of these alternative pathways could function to suppress tumorigenesis. We have previously shown that Wnt5a is required during embryonic development for the morphogenetic movements that extend the trunk and tail and for the growth of the limbs, face, gut and genitalia. We have recently obtained strong genetic evidence that indicates that Wnt5a signals through the Wnt/PCP pathway to regulate trunk and tail development, and that Wnt5a also has roles in neural tube closure and heart outflow tract development. Analysis of Wnt/beta-catenin lacZ reporters in Wnt5a mutants, together with the analysis of Wnt/beta-catenin target gene expression, has failed to reveal any evidence supporting a role for Wnt5a in the Wnt/beta-catenin pathway. Nevertheless, we have discovered that mutations in Wnt5a can enhance cancers of the immune system caused by mutations in components of the Wnt/beta-catenin pathway. Our data indicates that Wnt5a functions as a haploinsufficient tumor suppressor, suppressing T cell lymphoblastic leukemia possibly through the repression of Runx1. These genetic interaction studies have led to the development of a mouse model of T-cell lymphoma. To gain a mechanistic understanding of Wnt5a signaling, we have generated transcriptional profiles of Wnt5a mutant embryos. This approach identified 200 genes differentially expressed in wildtype and Wnt5a mutant embryos. Interestingly, there is virtually no overlap between the Wnt3a and Wnt5a target gene sets despite their overlapping embryonic expression. This suggests that the two Wnts utilize unique signal transduction pathways. Although these results are preliminary, we have identified an interesting gene, Ror1, which has been previously suggested to be a Wnt5a receptor, and which is upregulated by the absence of Wnt5a. This suggests a compensatory response in the responding cells to the lack of ligand. Characterization of this and other target genes is ongoing. Are Wnts the asymmetric cues that orient PCP along the AP body axis? If so, what are the mechanisms that translate these signals into the polarized cell movements of CE? Our transcriptional profiling of Wnt3a mutants outlined in project 1 indicated that potentially important PCP genes were downregulated. For instance, Wnt5b, which functions redundantly with Wnt5a (A. McMahon, unpublished), was strongly downregulated in the PS as assessed by microarray and WISH. Similarly, Prickle2 which is a core component of the PCP pathway and is asymmetrically distributed in the cochlea, is also downregulated in Wnt3a mutants. Intriguingly, Prickle2 was also down-regulated in our Wnt5a-/- profiles. Both Wnt3a and Wnt5a are expressed in the posterior end of the embryo, but Wnt5a mRNA is asymmetrically distributed in the mesoderm ie. graded with highest levels in the posterior PS. Our future experiments will distinguish whether canonical or noncanonical Wnts can impart AP polarity on the expression of core components of the PCP pathway. We have also identified mouse homologues of Daam (Dishevelled (Dsh)-associated activator of morphogenesis), a Formin protein that promotes actin polymerization and which interacts directly with Dsh and Rho to control convergent extension in frogs. We have generated numerous tools including targeted conditional loss-of-function alleles, LacZ-tagged alleles, tagged cDNAs and antibodies to understand how the Daam proteins function in the Wnt pathway to regulate cell and tissue polarity during mouse embryogenesis. We have found that our Daam1 loss of function mutants result in embryonic lethality at mid-gestation due to defects in the development of the placenta. We are continuing to characterize the mutant placentae. To gain a mechanistic understanding of Daam protein function, we have performed a yeast 2-hybrid assay with Myriad Genetics to identify Daam-binding proteins. This screen has been remarkably productive, identifying 65 directly interacting proteins that we are currently validating.